Despite the presence of a vigorous, broadly reactive HIV-1 specific immune response in infected individuals, progression to AIDS almost inevitable occurs. It has been postulated that mutations within critical epitopes may allow HIV-1 to escape immune recognition by cytotoxic T lymphocytes. A corollary to this hypothesis is that the ability of the host to generate new CTL responses may be a critical factor in containing viral replication. We have developed methods to sequence the T cell receptor genes of HIV-1 specific CTL and have shown that the vigorous CTL response in an infected individual may be due to a monoclonal or oligoclonal expansion of effector cells. We have also found instances in which HIV-1 variation within CTL epitopes leads to a loss of recognition by CTL isolated from that individual. Our ability to follow specific clonal responses by the analysis of TCR usage will allow us to determine the fate of these cells over the course of infection. The purpose of the experiments in this proposal is to use T cell receptor (TCR) sequence as a means to track specific CTL responses, and to develop potential methods of broadening the CTL response to a given epitope. Specifically we propose to 1) analyze the TCR repertoire of HIV-1- specific CTL. 2) compare the frequency of CTL clones as determined by TCR usage with the frequency determined by functional precursor frequency assays. 3) Use TCR gene sequences to track CTL clones over time, and compare with the magnitude of the CRL response as measured by functional precursor frequency assay. 4) evaluate techniques designed to expand the number and specificity of CTL that recognize particular epitopes. Research will focus on subjects for whom major CTL epitopes have been identified and from whom cryopreserved peripheral blood mononuclear cells and CTL clones of defined specificity are available. HIV-1 DNA corresponding to HIV-1 CTL epitopes will be amplified and sequences. Synthetic peptides representing these epitopes will be evaluated for mutations that allow for escape from CTL recognition. We will then evaluate the possibility of using autologous blood dendritic cells incubated with variant peptide to recruit new CTL responses in vitro. The TCR of CTL generated in this manner will be sequenced after polymerase chain reaction in order to correlate TCR gene usage with recognition of variant epitopes. The results of the research will further our understanding of how the TCR usage of HIV-1-specific CTL allows the host to respond to HIV-1 antigenic variation, and will help answer whether the clonal exhaustion of virus-specific CTL occurs over time. The in vitro expansion of HIV-1 specific CTL populations with broadened epitope specificity may lead to practical strategies for HIV-1 immunotherapy.